A Chemical Counterpart to the Resolution Step of Nature’s Intein-Mediated Protein Splicing

In the course of an attempted total chemical synthesis of the ant insulin-like peptide-2 (ILP2) protein molecule, specific cleavage of a backbone peptide bond in a branched ester-linked polypeptide chain with concomitant peptide splicing was observed. The side reaction was investigated in model compounds. Here, we postulate a chemical mechanism for this novel polypeptide backbone cleavage reaction as a chemical counterpart to the resolution step of biochemical intein-mediated protein splicing.


Analytical LC-MS
Analytical reversed phase HPLC-MS was performed using an Agilent 1100 series HPLC system equipped with an online MSD ion trap.Conditions for the chromatographic separations are provided under each chromatogram.Masses were obtained by online electrospray mass spectrometry.All MS data shown were collected across the entire principal UV absorbing peak in each chromatogram.

Preparative Reverse-phase HPLC purifications
Crude peptides and ligation reaction products were either dissolved in 6M GuHCl or in suitable solvent mixtures (maximum of 10% B), acidified to pH 2-3, and filtered (0.22μ).The clear solution was then loaded onto either a C4 (10x100 mm) in-house packed

Synthesis of Ser
Owing to high hydrophobicity and difficulty in handling and purification of Ser A1 -Tyr A21 -SCH2CO-Ala thioester, an Arg4-tag was incorporated in the thioester-leaving group in an optimized synthesis. 2Boc chemistry SPPS 'in situ neutralization' protocols as described above were carried out on Boc-Ala-OCH2-Pam-resin (0.2 mmol)).After assembly of the target sequence by SPPS, the final N α -Boc group was removed and the peptide was cleaved from resin with simultaneous removal of all the protecting groups with the exception of His(DNP), using 5% p-cresol/HF at 0 o C for 1 h.After careful evaporation of HF at 0 o C, the resulting residue was treated with ice-cold ether.After preparative HPLC as described in METHODS, purified peptide was obtained in 11% yield (72 mg, 22.2 µmol).
Following removal of the SerB28 Fmoc group, Boc SPPS cycles were carried out from Asn B27 through Thz B10 using the Boc chemistry 'in situ neutralization' protocols described above.After assembly of the target sequence by SPPS, the final N α -Boc group was removed and the peptide was cleaved from resin with simultaneous removal of all the protecting groups using 5% p-cresol/HF at 0 o C for 1 h.After preparative HPLC as described in METHODS, purified target peptide was obtained in 5.1% yield ( 63

S14
While these initial reaction conditions failed to produce any full-length material, the modified procedures described in main text METHODS significantly suppressed the cleavage reactions (Figure S9).Analysis of peak ratios between the peak at 24.8 min (compound 6b) and peaks at 27-28 min (compound 4, 5 or 6) clearly shows significant cleavages even under optimized reaction conditions (Table S1).

Stability of ester bond under LiOH saponification conditions
Support for the rearrangement of the once-cleaved ester-containing fragment was evident when the peptide Thz B10 -Ser B28 [O(Gly A23 -Cys A22 )]-Phe B30 (3) was treated under 25 mM LiOH (pH ~12) saponification conditions in water at 4 o C. The cleavage reaction was rapid in this case.Two main products Cys A22 -Gly A23 -Ser B28 -Met B29 -Phe B30 and Thz B10 -Asn B27 -OH were formed presumably due to the base catalyzed cleavage at the -Asn B27 -Ser B28 [O(Gly A23 -Cys A22 )] bond, followed by two other reactions: rapid intramolecular nucleophilic attack by the Ser B28 alpha amino group on the ester bond carbonyl of Gly A23 , to give the amide linked product, stable to hydrolysis; and, rapid base catalyzed hydrolysis of the C-terminal B27 aspartimide, to give the Thz B10 -Asn B27 -OH.Such a series of reactions would explain why the ester bond initially believed to be present in the Cys A22 -Gly A23 -Ser B28 -Met B29 -Phe B30 cleavage fragment did not hydrolyze under these extremely basic conditions.

Folding of ant ILP2 full length depsipeptide
Folding of the crude full-length depsipeptide was carried out in an analytical scale under the following conditions, which were based on the conditions used to folded human ester insulin: 3 1.5 M GuHCl, 20 mM Tris, 8 mM Cysteine, 1 mM Cystine, pH 7.6 at 4 o C (Figure S12) and room temperature (Figure S13).In both of the conditions folding was complete within 1 h, however better results were observed in 4 o C. Cleavage at the Asn B27 -Ser B28 site was also seen under the folding conditions.

Figure S1 .
Figure S1.Analytical LC-MS data of Fmoc-Ser(OtBu)-OPac. (Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 20 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of the main UV peak.

Figure S2 .
Figure S2.Analytical LC-MS data for Fmoc-Ser(OH)-OPac.(Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 20 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks a-c.

Figure S3 .
Figure S3.Analytical LC-MS data for Fmoc-Ser[O(Boc-Gly)]-OPac. (Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks a-b.

Figure S4 .S9 5 . 3 1 )
Figure S4.Analytical LC-MS data for Fmoc-Ser[O(Boc-Gly)]-OH. (Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks a-b.

Figure S5 .
Figure S5.Analytical LC-MS data for Ser B1 -Tyr B9 -SCH2CO-Ala.(Top panel) Reverse phase HPLC -The chromatographic separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks.Peak a: deletion of Ala and -SCH2CO moieties (-145 Da)].

Figure S6 .
Figure S6.Analytical LC-MS data for purified Ser A1 -Tyr A21 -SCH2CO-Arg4-Ala.(Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks.Synthesis of Thz B10 -Ser B28 [O(Gly A23 -Cys A22 )]-Phe B30 (3) mg, 25.6 µmol).Upon removal of the N-alpha Fmoc of SerB28, an internal nucleophilic attack by the SerB28 alpha-amine on the ester moiety, favored by a five-membered ring geometry, would lead to the terminated peptide CysA22-GlyA23-SerB28-MetB29-PheB30 following the final global deprotection and cleavage.We did not observe such a byproduct, but on the other hand we were not explicitly looking for it and the suggested side reaction may have contributed to the low overall yield of the purified branched depsipeptide segment.

Figure S8 .
Figure S8.A) Analytical LC-MS data for Synthesis of Ant insulin full-length peptide.(Top panel) Reverse phase HPLC -The chromatographic separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks.B) Schematic structures of the observed products.

Figure S9 .
Figure S9.A) Analytical LC-MS data for Synthesis of Ant insulin full-length peptide using modified conditions.(Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks.B) Schematic structures of the observed products.

(Figure 10 )Figure S10 .
Figure S10.Analytical LC-MS data for Purified Ant insulin full length polypeptide.(Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks.

Figure S11 .
Figure S11.Analytical LC-MS data for LiOH saponification of Thz B10 -Ser B28 [O(Gly A23 -Cys A22 )]-Phe B30 (3) at 4 o C. (Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−28%) of solvent B in solvent A over 23 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks.

Figure S12 .
Figure S12.Analytical LC-MS data for folding of Ant insulin full length polypeptide at 4 o C. (Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 20 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks.

Figure S13 .
Figure S13.Analytical LC-MS data for folding of Ant insulin full length polypeptide at room temperature.(Top panel) Reverse phase HPLC separations were performed on a C4 (4.6x150 mm) column at 40 o C, using a linear gradient (5−45%) of solvent B in solvent A over 20 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectra, taken across the whole of each of the UV peaks.

Figure S14 .
Figure S14.LC-MS data for the synthesis of Ac-Phe-Arg-Ala-Asn-Ser(OAc)-Phe-Arg-Ala.(Top panel) Reverse phase HPLC chromatographic separations were performed on a C8 (4.6x150 mm) column at 40 o C, using a linear gradient (1−41%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectrum taken across the whole of the main UV peak.

Figure S15 .
Figure S15.LC-MS data for the stability studies of Ac-Phe-Arg-Ala-Asn-Ser(OAc)-Phe-Arg-Ala.(Top panel) Reverse phase HPLC chromatographic separations were performed on a C8 (4.6x150 mm) column at 40 o C, using a linear gradient (1−28%) of solvent B in solvent A over 27 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectrum taken across the whole of the main UV peak.

Figure S16 .
Figure S16.LC-MS data for the synthesis of Ac-Phe-Arg-Ala-Asn-Ser(O-Gly)-Phe-Arg-Ala.(Top panel) Reverse phase HPLC chromatographic separations were performed on a C8 (4.6x150 mm) column at 40 o C, using a linear gradient (1−41%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectrum taken across the whole of the main UV peak.

Figure S17 . 13 .
Figure S17.LC-MS data for the synthesis of Ac-Phe-Arg-Ala-Asn-Ser(O-Gly-Ac)-Phe-Arg-Ala.(Top panel) Reverse phase HPLC chromatographic separations were performed on a C8 (4.6x150 mm) column at 40 o C, using a linear gradient (1−41%) of solvent B in solvent A over 40 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectrum taken across the whole of the main UV peak.

Figure S18 .
Figure S18.LC-MS data for the stability studies of Ac-FRANS(O-Gly)FRA and Ac-FRANS(O-Gly-Ac)FRA at pH 7.0 with and without TCEP.(Top panel) Reverse phase HPLC chromatographic separations were performed on a C8 (4.6x150 mm) column at 40 o C, using a linear gradient (1−31%) of solvent B in solvent A over 30 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectrum taken across the whole of the main UV peak.

Figure S19 .
Figure S19.LC-MS data for the stability studies of Ac-FRANS(O-Gly)FRA and Ac-FRANS(O-Gly-Ac)FRA at pH 5.5 with and without TCEP.(Top panel) Reverse phase HPLC chromatographic separations were performed on C8 (4.6x150 mm) column at 40 o C, using a linear gradient (1−31%) of solvent B in solvent A over 30 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectrum taken across the whole of the main UV peak.

Figure S20 .S30 14 .
Figure S20.LC-MS data for the stability studies of Ac-FRANS(O-Gly)FRA and Ac-FRANS(O-Gly-Ac)FRA at pH 8.0 with and without TCEP.(Top panel) Reverse phase HPLC chromatographic separations were performed on a C8 (4.6x150 mm) column at 40 o C, using a linear gradient (1−31%) of solvent B in solvent A over 30 min (solvent A = 0.1% TFA in water, solvent B = 0.08% TFA in acetonitrile) at a flow rate of 1.0 mL/min with detection by UV absorption at 214 nm.(Bottom panel) Online ESI-MS spectrum taken across the whole of the main UV peak.

Table S1 .
Time course of cleavage products during one-pot native chemical ligation steps.